Mycoplasma contamination is one of the most difficult cell culture problems to recognize because infected cultures may appear completely normal. Mycoplasmas are very small, cell-wall-deficient bacteria that can persist in cell cultures without producing the obvious turbidity typically associated with bacterial contamination.
Despite their subtle appearance, mycoplasmas can alter cell proliferation, metabolism, gene expression, membrane properties, transfection efficiency, and other experimental readouts. Routine mycoplasma detection is therefore an important component of cell culture quality control.
What Is Mycoplasma and Why Is It a Problem?
Mycoplasmas are among the smallest self-replicating prokaryotic organisms, with many species measuring roughly 0.3–0.8 µm. They lack the peptidoglycan cell wall found in many bacteria.
This creates several practical problems.
First, infected medium may remain visually clear even when substantial numbers of organisms are present. Second, antibiotics that act on bacterial cell-wall synthesis, such as penicillin, are ineffective because mycoplasmas lack the relevant target.
Mycoplasma contamination can therefore remain undetected for multiple passages while spreading to other cultures.
How to Detect Mycoplasma Contamination
PCR-Based Detection
PCR is widely used for mycoplasma detection because it can identify mycoplasma nucleic acids with high sensitivity and relatively rapid turnaround.
A positive result should be interpreted according to the assay's controls and validation criteria. Because nucleic-acid assays can potentially detect DNA from nonviable organisms, follow-up testing may sometimes be useful after treatment.
DNA Staining
Fluorescent DNA-binding stains such as Hoechst or DAPI can reveal small extranuclear DNA-containing particles around cultured cells.
This method can provide useful visual evidence, but interpretation requires experience and may be less sensitive than validated molecular assays.
Immunological Detection
Antibody-based methods, including enzyme-linked immunoassay approaches, can detect mycoplasma-associated antigens. Performance depends on the organisms recognized by the assay, so these approaches should be used according to a validated laboratory method.
Culture-Based Detection
Microbiological culture can determine whether viable mycoplasma organisms are present. The major disadvantage is that cultivation can take considerably longer than rapid molecular methods.
Culture-based testing can nevertheless be useful when viability information is important.
Methods for Eliminating Mycoplasma from Cultures
Discard and Restart When Possible
For replaceable cultures, the preferred response is generally to discard the contaminated culture and restart from a verified mycoplasma-free stock. This reduces the risk that residual organisms or contamination-induced cellular changes will compromise future experiments.
Consider Treatment Only for Irreplaceable Material
If the culture is rare, unique, or impossible to replace, mycoplasma elimination may be attempted under a dedicated laboratory protocol.
Antimicrobials with activity against mycoplasma include selected agents from the quinolone, tetracycline, and macrolide classes. Ciprofloxacin is one example of a quinolone that has been evaluated for this purpose. However, susceptibility varies, resistance can occur, and treatment can be toxic to the cultured cells.
Treatment should therefore follow a validated eradication protocol rather than an improvised antibiotic concentration or duration.
After treatment, cultures should remain quarantined until repeated testing confirms that contamination has been eliminated.
Preventing Mycoplasma Contamination in the Lab
Quarantine Incoming Cell Lines
Treat newly introduced cultures as potentially contaminated until screening is complete.
Screen Routinely
Perform mycoplasma testing at defined intervals and at important experimental checkpoints, such as before cell banking or critical assays.
Limit Shared Reagents
Avoid inserting the same pipette or transferring material between multiple cultures in ways that could spread contamination.
Practice Good Aseptic Technique
Careful handling remains the most important preventive measure. Standard antibiotics cannot be relied upon to prevent mycoplasma contamination.
FAQ
Common questions
Can I see mycoplasma under a normal microscope?
Usually not reliably. Infected cultures often look normal under routine microscopy.
Does penicillin eliminate mycoplasma?
No. Mycoplasmas lack the cell wall targeted by penicillin.
Should I treat or discard an infected culture?
Discarding and restarting is generally preferable when a clean replacement exists. Treatment is mainly considered for valuable cultures that cannot reasonably be replaced.
How do I know treatment worked?
Retest using a validated mycoplasma detection method after the treatment and recovery period. Follow your lab's standard verification protocol.
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